An analysis of the ‘tolerance’ which develops to analgetic electrical stimulation of the midbrain periaqueductal grey in freely moving rats

Brain Research. 435 ( 1987197- I I I Elsevier

97

BRE 13085

An analysis of the 'tolerance' which develops to analgetic electrical stimulation of the midbrain periaqueductal grey in freely moving rats M.J. Millan, A.

Cztonkowski*

and A. Herz

Deparonent of Neuropharmacology. Max-Planck-h~stitutfiir Psychiatric. Planegg-Martb~sried f F. R. G. ) (Accepted 12 May 1987)

Key words: Periaqueductal gray; Opioid: Opioid receptor: Pain; Nociception; Tolerance: Conditioning

Electrical stimulation t~l the ventral midbrain periaqueductal grey (PAG) elicits an opioidergic antinociception against noxious heat and pressure in freely moving rats. Recurrent stimulation was associated with a gradual decline and eventual loss of this stimulationproduced antinociception (SPA). This could be reinstated by an increase in current intensity and this reinstatement was preventable by naloxone. The current intensity-antinociception (dose-response) curve was shifted to the right in recurrently stimulated rats and parallel to that in naive animals. The loss of SPA upon repetitive simulation did not represent a conditioning phenomenon. Thus. tolerant rats exposed to all cues which accompanied stimulation revealed no (compensatory) hyperalgesic response - - but rather a slight antinocieeption. Further, SPA recovered spontaneously ill tolerant rats. Moreover, 'extinction" by repeated exposure to all cues accompanying stimulation did not restore or accelerate the recovery ol"SPA in tolerant animals. Tolerant rats sho:~ed no depletion in midbrain PAG or other CNS or hypophyse:d pools of fl-endorphin, Met-enkephalhl or dynorphin indicating that a depletion of endogenous optold peptides does not underlie the tolerance which develops to stimulation. In fact recurrently stimulated rats did not show any of the pronounced effects upon CNS pools of opioid peptides which are seen with long-term stress. Moreover, repetitively stimulated rats revealed no indications of stress as judged by a diversity of stress-sensitive parameters; basal nociccptive th,'eshold, core te~nperature. ingestive behaviour, body weight, adrenal weight and hypophyseal secretion offl.endorphin and prolactin. The data offer two major conclusions. Firstly, the gradual loss of analgesia upon recurrent stimulation of the midbrain PAG does not reflect a generalized debilitation or stres~ and neither a conditioning phenomenon nor a depletion of pools of endogenous opioid peptides. R,ither it closely corresponds to the pharmacological definition of tolerance and may reflccl a process occurd,~g at the level of the opt, id receptor and coupled processes. This finding explains the cross-tolerance which we observe recurrently stimulated rats to dis!,!a? ~ ,norphine. Secondly, this SPA is ,~ot a form of stressoinduced an:dgesia and rats undergoing recurrent stimulation reveal no i,ldications of stress as judged by biochemical, physiological .'rod behaviot, r:d parameters.

INTRODUCTION In both restrained and freely movi~g rats, electrical or chemical stimulation of discrete regions of the brainstem reliably elicits an antinociception (analgesia) referred to as stimulation-produced analgesia (SPA) 5"7"25"34"35"37"40"41'43.O f particular interest is the SPA e v o k e d from the ventral midbrain periaqueductal grey ( P A G ) in the region of the dorsal raph6 which appears to be m e d i a t e d by an opioidergic

mechanism 7'43'5'~. O u r recent work has indicated that it may b e / 3 - e n d o r p h i n which is the opioid peptide, and the Ft-receptor which is the opioid receptor type, underlying the SPA elicited from the ventral midbrain P A G 4t~'~3'4~. in view of the involvement ot opioid systems, one might anticipate that a tolerance may develop upon recurrent stimulation, in analogy to the tolerance which results upon r e p e a t e d administration of opioid agonists such as m o r p h i n e or fl-endorphin. Indeed, it

* Permaneni. dddress: Department of Pharmacology, Institute of Physiological Sciences. Medical Academy of Warsaw. Poland. Correspondence: M.J. Millan. Department of Neut,.,pharmacology, Max Planck-lnstitut fiir Psychiatrie, Am Klopfcrspitz 18a, D8033 Planegg-Martinsried. F.R.G. 0006-8993/87/$03.50 © ! 987 EEevier Science Publishers B.V. (Biomedical Division )

98 has been reported that the magnitude of SPA declines upon recurrent stimulation5"35"37'38'4°. However, tolerance is a strictly and carefully defined phenomenon and does not simply correspond to a loss of response. By tolerance, it is understood that the original effect can be restored by an increase in stimulus intensity and that there is a shift in the dose-response curve to the right. In addition, it is pre-supposed that the restored effect is mediated by an identical (in this case, opioidergic) mechanism. Moreover, it is clear that there are a number of mechanisms which could underlie a loss of the analgetic effects of PAG stimulation. For example, (i) the animals may be debilitated and manifest generalized deficits or signs of chronic stress, (ii) there may a depletion of the endogenous opioid which mediates SPA, or (iii) tolerance may reflect a conditioning phenomenon and, in ace 9rdance with a pavlovian-like model, a conditioned opponent response (see Discussion) may be acquired. Thus, a major purpose of this study was to address the issues of whether a tolerance does indeed develop to the SPA elicited from the PAG and, if so, what may be the mechanism underlying this. In addition, a number of authors have drawn parallels between SPA and the analgesia induced by stress and it has even been speculated that SPA might represent a type of stress-induced analgesia ~,14,15,29,'a~', ~t:2,sl. The use of freely moving animals has allowed for a refutation of this contention in demonstrating that the effects of acute stimulation may be clearly distinguished from those of stress by a diversity of behavioural, physiological and biochemical parameters reflecting the activity of opioidergic and other substrates 4°:1:3, In the present study, complementary data are presented concerning recurrent stimulation which shows that the effects of this may clearly be differentiated from those of chronic exposure to stress. MATERI ALS AND METHODS

Surgery Male Sprague-Dawley rats of 20C-250 g were allowed unlimited access to rat chow and water and were individually housed in a temperature (22 °C) and humidity (60%) controlled room, Lights were on frora 7.30 to 19.30 h. All experiments were performed within the light phase. The procedure employed was exactly as that de-

scribed previously4°'43'4'~. Briefly, a single, bipolar, stainless-steel electrode was implanted under pentobarbital anaesthesia (50 mg/kg) into the ventral midbrain PAG of rats mounted in a stereotaxic apparatus. Coordinates, according to the atlas of K6nig and Klippel3° were, with respect to the interauricular line: h~rizontal +0.5, vertical +4.4, lateral -0.2 mm. To avoid damage to the sinus on the surface e.f the brain, electrodes were implanted at a lateral angle of 15°. Following implantation, rats were allowed 7 days for recovery during which time they were adapted to handling and gentled. Unless otherwise stated, all experiments were performed on independent groups of rats.

Stimulation The procedure for stimulation was as that described previously4°'43"44. Rats were placed in a modified Skinner box and the electrode was attached to a cable which was in turn connected to a wheel in the roof oi' the chamber which led to the stimulator. This set-up allows for the completely free behaviour of the rat during stimulation. Rats were stimulated for 10 rain as previously (constant-current, biphasic, rectangular pulse-pairs; pulse duration 50 #s, inter-pulse interval 10/~s and one train of 425 ms/min). Unless otherwise specified, tile current intensity employed was 350~A: this was attained by a gradual increase in current iLatensity over the first minute of stimulation. Nociceptive testing Nociceptive thresholds were determined as previously4°'43-45 immediately prior to and following stimulation. Thresholds to heat were always determined prior to thresholds to pressure. Tail.flick test to heat. The threshold for removal of the tail from a noxious beam of light directed at its tip was evaluated. Rats were gently restrained under paper wadding and the mean of 3 values (with consecutive values separated by an interval of 10 s) was taken. Beam intensity was such that for all experiments basal thresholds of ca, 3 - 4 s were acquired. A cut-off of 8 s was imposed in order to preclude tissue damage. Withdrawal test to pressure. An incremental pressure was applied via an automated, wedgeshaped piston to a point 2 cm from the tip of the tail of gently restrained rats. The pressure at which the tail

99 was withdrawn was determined. The mean of 3 readings (separated t~y intervals of 10 s) was determined. ~asal values were around 120 g and a cut-off of 400 g was enforced to ensure that no tissue damage ensued.

Evaluation of rat behaviour during stimulation In the course of turning up the current for stimulation, the current intensity at which locomotor effects were initially seen to commence was recorded. A previously characterized 4°'43 simple scoring system was used for the semiquantitation of this locomotor behaviour: 0 = no overt effect, 1 = turning movement of the head, 2 = mild rotation of the whole body, 3 = strong rotation of the whole body. Core temperature was monitored immediately prior to and immediately following stimulation, in each case just after determination of nociceptive thresholds. A digital thermistoprobe was inserted to a depth of 5 cm into the rectum of gently restrained rats for 30 s and the temperature read off the scale.

Development of'tolercmce' to stimulation Rats were subdivided into 3 groups termed recurrent (REC), single (SNG) and chamber (CHA) respectively; they were treated as follows. REC rats were repetitively stimulated twice a day as detailed above for a total of 7 sessions, that is three and a half days (morning, afterqoon, morning, etc.). C H A rats were treated identically with the exception that no current was delivered on any occasion. SNG rats were also treated in this wa 3, and current was only delivered on the final (seventh) test session. Antinociception, the change in core temperature and locomotor effects were recorded across all sessions, as described above. In an independent group of animals, experiments were performed to quantify the degree of 'tolerance" developed to the antinociceptive effects of stimulation. In this study, both REC and CHA groups were treated as described above over 7 sessions. On a further (eighth) session, rats were stimulated with varying current intensities. Thus, a current intensity-antinociception (equivalent to dose-response) curve was determined for each group. In a further experiment, it was determined whether (in analogy to naive rats) the antinociception which can be reinstated in 'tolerant' rats was suscept-

ible to antagonism by the opioid antagonist, naloxone. Rats were recurrently stimulated over 7 sessions as described until '=tolerance' had developed. They were then implanted subcutaneously on the flank with an osmotic minipump. This contained either water or a solution of naloxone of 100 mg/ml and delivered its contents at a rate of 1 ld/h: this corresponds to a dose of 0.5 mg/kg naloxone/h. In extensive previous work, we have shown this dose to selectively antagonize/~-opioid receptors 43 (Millan and Morris; Shippenberg and Millan, submitted). In order to confirm the efficacy of pumping, body weight in addition to food and water intake was monitored for the 24 h following implantation. On the following day (the eight ression), current intensity was increased to 900/~A and its antinociceptive effects evaluated in rats receiving naloxone and their control counterparts.

Examination of conditioning effects In this experiment, ,REC and SNG rats were treated exactly as described above for 7 sessions over the course of which the analgetic effect of stimulation declined. On the eighth session both REC and SNG rats were divided into 3 groups as follows. In the first, nociceptive thresholds were determined, the rats returned home for 10 min, then thresholds re-evaluated. in ~he second, nociceptive thresholds were measured, the rats stimulazed for 10 rain as on the previous sessions, and thresholds re-measured thereafter. In the final group, nociceptive thres]:o!d~ were determined, rats placed in the chamber but not stimulated and thresholds thea retaken. In the case ot this final group, they were re-tested in this manner for a further 3 sessions, over the next one-and-a-half days. In a further experiment, REC rats which had developed •tolerance' to stimulation over 7 sessions were divided into two groups and examined as follows. They were either allowed to rest in their home cages for 5 days or they were placed twice daily in the chamber without being stimulated: that is, for a total of 10 sessions of 10 rain each. On each occasion, core temperature and nociceptive thresholds were determined before and after placement in the chamber. After these 10 sessions, in a final session, the antinociceptive effects of stimulation were re-evaluated in both groups of rats employing the usual procedure. Finally, for a further 5 days, all rats were allowed to

100 rest in ti~eir home cages, whereafter the effects of stimulation were re-determined.

Evaluation of influence of recurrent stimulation upon physioiogical parame,ers and opioid peptide systems Rats were divided into 4 groups termed recurrent (REC), single (SNG), chamber (CHA) and home (HOME). REC rats were stimulated for a total of 4 sessions at a current intensity of 300/tA. From the fifth session onward, until a total of 14 sessions over 7 days. current intensity was increased step-wise (see Fig. 6) in order to maintain its analgesic efficacy. Antinociception was determined on each day of stimulation. Rats in the SNG group were treated likewise with the exception that only on the final (14th) session was current delivered. Rats in the CHA were also treated in this way except that on no occasion was any current delivered. Rats in the HOME group were simply allowed to rest in their home cages. Immediately prior to the 14th session, the basal nociceptire thresholds and core temperature of HOME rats were taken. I'hroughout this testing period, body weight, daily food and water intake were monitored on all groups of rats. On the day following completion of testing, all rats were sacrificed by decapitation. The adrenal glands and the iumbosacral spinal cord were rapidly removed, the pituitary divided into constituent anterior and neurointermediate lobes, trunk blood collected and the brain dissected to yield the midbn, in PAG, striatum, meduUa.pons, thalamus, septum and hypothalamus. The midbrain PAG incorporated the region within which our electrode tips are located and included both the ventral and dorsal parts of the PAG. The piece taken was approximately rectangular in shape and consisted of a block 1.5 mm on each side of and 2.0 mm dorsal and ventral to the ventricle: it weighed approximately 100 rag. All proceoures for treatment of tissue, extraction of peptides and radioimmunoassay of opioid peptides have been extensively detailed elsewhere. The antibody characteristics have also been thoroughly described previously9'22'~'45. Briefly, the antiserum to fl-endorphin (fl-EP) recognized fl-lipotropin to an equimolar degree but showed negligible recognition of ACTH, Met- and Leu-enkephalin, dynorphinl_t7 A (DYN) and all other peptidcs tested. That to DYN failed to cross-react to DYN B, DYNI_8, a- or fl-neoendor-

Fig. 1. Schematic representation of electrode tip locations within the ventr,'d periaqueductal grey. derived from a coronal section through the brain. The cross-hatched area is that within which the electrodes were located.

phin, Leu- and Met-enkephalin, heptapeptide, octapeptide and fl-EP. That to Met-enkephalin showed less than 1% cross-reactivity to Leu-enkephalin and none to the above-mentioned peptides. The detection limits for fl-EP, DYN and Met-enkephalin were, respectively, 2, 1 and 20 fmol/assay tube. Prolactin (PRL) was measured by materials kindly provided by the NIADDK, National Institute of Health, Bethesda, MD, U.S.A. The standard was rat PRL RP2. The detection limit was 0.05 ~g/ml plasma.

Histology Upon completion of testing, rats were decapitated, their brains removed, frozen on dry-ice and 2 0 / , n thick coronal sections cut on a cryostat: these were stained with Cresyl violet for viewing. In addition, some sections were stained for glial fibrillary acidic protein,

Statistics Student's two-tailed t-test, the t-test for paired samples and analyses of variance (ANOVA) of appropriate design were employed as indicated l,elow. Since the data for antinociception represented percent values, these were normalized by an arc-sin transformation prior to ANOVA. Following one-way ANOVA, Student-Newman-Keuls (SNK) test for multiple comparisons was applied: the level of significance was set for this at P ~< 0.05. These analyses may be found in the Results. In addition, since in certain cases we had hypothesized differences between particular pairs of groups prior to experimentation, it

1(11

was valid to compare these by use of Student's twotailed t-iest; these analyses are given in the legends to the Figures. Linear regressions were determined for the dose-response curve presented in Fig. 3 and the slopes of these curves calculated and compared by the Method of Least Squares. RESULTS

Effects of stimulation: development of'tolerance' The electrodes were reproducibly located in all

. a,

groups: they were placed (as fully detailed in our previous s t u d i e s "m.'~3-'~4) in the ventral midbrain PAG in and lateral to the dorsal raphC Fig. 1 is a schematic summary of electrode placements. In all animals the electrode tract was clearly visible and an area of gliosis was apparent in the immediate vicinity of the electrode. There was no other sign of neural damage. The effects of stimulation were very similar amongst all groups and have been characterized in detail in our previous w o r k 4°-43.44. In addition to the induction of an antinociception against both noxieus

Antinociception

b,

It

175 '

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Change in Core Tc,nperature

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d. Locomotor Threshold ~AMP 200.

20'

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4oJ TEST SESSION

Fig. 2. The inl'luencc of recurrent stJnlul;alJon of the midbr:lin pcriaqueductal grey upon noeiceptive thresholds, core lempcraturc and locomotor behaviour in the r;.it. In panel a, antinociception is expressed as a percentage of basal pre-stimulation values (defined as IOllq- ): in panel b, the change in core temperature (post- vs pre-stimulation) is presented: in panel c, the Jntensily Of Ihe locomotor elfeels is shown and in panel d, the stimtdation current at which locomotor effects initially appeared is depicted. Symbols as follows. For panel a, for rats stimulated on each session (O = heat and O -- pressure), for rats stimulated only on the final session ( A = heat and ,~\ = pressure), for rats stimulated on no sessions (in = heal and [] = pressure). For panels b - d , rats stimulated on each occasion (Q). rats stimulated only on the final sessions (,~,) and rats not stimulated on any session (in). For further details, see Methods. "Fherc ~as no significant difference between the basal nociceptive thresholds and basal core temperature (pre-stimulation) amongst tile various groups on any test se',~sion. Mean _ S.E.M.s depicted. For recurrently stimulated rats. n = 12. For rats stimulated only on the final session, n = 5 and rats not stimulated on any session, n = 8. Asterisks indicate significance of differences between values on session 7 :is compared :o session 1. (**P ~ {L0()I, paired t-test.) The crosses indicate the significance of differences between rats slimulatcd only on s~-ssion 7 and those which had been rendered tolerant by recurrent stimulation. '~P ~ 0.01}l (Student's two-tailed t-tesl).

1(12 pressure and heat, an elevation of core temperature and locomotor effects (which consisted of a rotation ipsilateral to the electrode) were seen (Fig. 2). Placement in the chamber in the absence of stimulation also elicited a hyperthermia but neither an antinociception nor locomotor effects (Fig. 2). On the first test session, for antinociception to heat: F2,19 = 17.94, P < 0.001; for antinociception to pressure: ~.m = 15.24, P < 0.001. A significant difference was seen between REC and CHA/SNG groups for both heat and pressure upon SNK analysis. There was no significant difference between the various groups as regards the rise in core temperature; F2.m = 0.62, P > 0.05. As may be seen from Fig. 2, with repeated stimulation the ntagnitnde of the antinociception evoked progressively diminished. That this effect did not merely reflect recurrent exposure to the chamber is indicated by the fact that rats in the SNG group which were stimulated only on session 7 developed a full antinociception. On session 7, for heat, F2.t9 = 18.77, P < 0.001 and for pressure, F2.t9 = 16.66, P < 0.001. For both heat and pressure, on session 7 SNG rats were significantly different from REC/CHA rats upon SNK analysis. In contrast to antinociception, there was no alteration in the hyperthermic effects of stimulation upon repetitive testing (Fig. 2). Whereas there was likewise no alteration in the intensity of the locomotor effects evoked, the threshold for the initiation of these gradually rose across ~uccessive test sessions (Fig. 2).

Dose-response relationship in 'tolerant' rats: naloxone reversal of reinstated antinociception Although the data in Fig. 2 suggest the development of tolerance, the most important criterion of this is a shift in the dose-response (current intensity-antinociception) relationship to the right. An increase in current intensity should restore the antinociceptive effect of stimulation. Fig. 3 reveals that this is indeed the case. The equations of the curves presented in this figure were computed to be as follows. Naive, heat y = 89.82 + 0.17x; naive, pressure y = 89.24 + 0.16x" "~lerant, h e a t y = 53.22 + 0.13x, and tolerant, pressure y -- 57.06 + 0. !3x. There was no significant difference between the slopes of these curves for naive as compared to tolerant rats as regards either heat or pressure (P > 0.05). Further, a

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Fig. 3. The antinociceptive influence of stimulation of the midbrain periaqueductal grey at various current intensities in 'naive' (hitherto unstimulated) and "tolerant' (recurrently stimulated) rats: reversal of reinstated antinociception in tolerant rats by naloxone. For further details, see Methods. Mean depicted (S.E.M.s are omitted for clarity). Asterisks indicate significanceof naloxone vs control values. ***P ~<0.0l (Student's two-tailedt-test). For ANOVA and equations of the curves, see Results.

multivariate ANOVA was performed on the data points acquired by use of current intensities of 350 and 600 ~tA (common to both naive and tolerant rats). There was a significant effect of current intensity: heat, Ft.2t -- 13,28, P < 0.001; pressure, Ft,,,.t = 27.21, P < 0.0(11. There was also a significant effect of tolerance: heat, Fi,2t -- 24.28, P < 0.001, and for pressure, F~.2t - 49.19, P < 0.001. in line with the lack of a significant alteration in slope, there was no significant interaction between current intensity and tolerance: heat, Ft,2, = 0.45, P = 0.51, and pressure, FI,21 = 0.59, P = 0.41. Treatment of rats with naloxone via osmotic minipumps produced the anticipated reduction in rate of body weight gain, food intake and water intake refleeting a blockade of endogenous opioid systems and demonstrating that the pumps were operating effectively (Table I). There was no influence upon basal nociceptive thresholds prior to stimulation (Table I). In iolerant rats receiving water via the pumps, an increase in current intensity to 900/~A reinstated the antinociceptive effects of stimulation (Fig. 3). However, in rats treated with naloxone, this antinociception was almost totally abolished (Fig. 3) indicating that this reinstated antinociception is opioidergic in nature.

103 TABLE [

The influence of naloxone perfi~sed via minipumps upon basal nociceptive thresholds and ingestive behaviour Mean + S.E.M. given. Body weight gain and intakes for 24 h following implantatioa of pumps.

Water (n =

4)

Naloxone (n = 5)

Fail-flick heat (x)

Tail-withdrawal pressure (g)

Body weight gain (g)

Food imake (g)

Wa,Lr (g)

3.74 + 0.15

7.92 + 0.52

+8.69 _+. 1.64

22.32 + 0.59

29.35 + 1.35

4.03 + 0.16

8.36 __.0.65

+2.14 ___1.05"

18.05 + 0.87*

20.65 _+0.71"*

.......

Asterisks indicate significance of naloxone vs water differences: *P ~<0.02, **P ~<0.005 (Student's two-tailed t-test).

Analysis of the role of conditioning in the developmem

antinociception" in contrast, recurrently stimulated rats which were re-stimulated or returned to their home cages for an identical period of time showed no such antinociception (Fig. 4). For heat, F,H = 12.17,

o f tolerance

Exposure of recurrently stimulated rats to the chamber in the absence of stimulation led to a mild

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TEST SESSION 8 Fig. 4. Cues associated with stimulation elicit a mild antinociception in rats rendered tolerant thereto. 'Naive' signifies rats which had been exposed to all cues of stimulation but not actually stimulated for 7 preceding sessions. 'Tolerant' refers to rats which had been stimulated on all preceding sessions. On session 8, oasal thresholds were determined and rats were either returned home for 10 min (HOME), placed in the stimulation chamber and not stimulated (CHA) or stimulated (STIM). The inset depicts the effect of re-exposure to the chamber without stimulation for a further 4 sessions in the group, tolerant/CHA which had shown a mild antinociception on session 8. For further details, see Methods. Mean + S.E.M. depicted, n ~>6 per column. Asterisks refer to the significance of: (i) naive/STIM vs naive/CHA; (ii) tolerant/CHA vs naive/CHA; (iii) tolerant/STIM vs naive/STIM. **P ~< 0.01, ***P ~< 0.001 (Student's two-tailed t-test). For ANOVA, see Results.

104

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TEST 9

HOME

Fig. 5. Re.exposure to all cues acc-3mpanying stimulation {'extinction') does not accelerate the recovery from tolerance to the antinociceptive effects of stimulation', this recovers spontaneously. Rats were stimulated for 7 sessions; they were then allowed either to re~t in their home cages for 5 days or were re.exposed to all cues accompanying stimulation twice a day for 5 days. They were re-stimulated on session 8. All rats were thereafter allowed to rest at home for a further 5 days whereafter they were again re-stimulated on sessior~9. For further details, see Methods. Mean +_S.E.M. shown, n ~ 7 per column, Asterisks refer to significance of differences between the followinggroups: (i) session 7 vs session 1: (it) session 8 vs session 7; (iii) session 9 vs session 8, *P <~0,05, **P <~0.Ol and ***P ~ 0,00! (paired t-test). For ANOVA, see Results,

P -- 0.003, and for pressure, F2,22 = 4.77, P = 0.019. In contrast, in naive (as yet unstimulated) animals (Fig. 4) and rats which had been stimulated only once previously (not shown), exposure to the chamber did not elicit an antinociception. In these naive animals, stimulation was effective. For heat, F2js = 43,13, P < 0,001, and for pressure, F2.18 = 43.13, P < 0.001, The inset to Fig. 4 reveals that this antinociception seen upon introduction of 'tolerant' rats to the chamber rapidly decayed with repeated exposure t h e r e t o In Fig. 5, the lack of influence of 'extinction' trials upon 'tolerance' to stimulation-evoked antinodception is illustrated; in addition, that 'tolerance' is lost and the efficacy of stimulation spontaneously recovered may be seen. Recurrent stimulation for 7 ses-

sions led to the familiar loss of analgesia. Following a rest period of 5 days, in which rats were either allowed to rest in their home cages or exposed to the chamber in the absence of stimulation twice a day, there was a slight recovery. The magnitude of this recovery did not differ significantly between the group receiving these extinction trials and those resting at home. Following a further 5 days of rest, a further recovery was seen though a complete restoral of the antinociceptive efficacy of stimulation was not apparent. There was a significant difference between the antinociception evoked in test session 9 vs test session 1 for both heat (P ~ 0.01) and pressure (P 0.05) in the paired t-test.

1(15 TABLE II

The influence of recurrent electrical stimulation of the midbrain periaquedactal grey upon selectedphysiological and beharioural parameters in the rat 'Home" signifies animals which remained in their home cages; "chamber', animals which were placed in the stimulation chamber twice a day for a week but not stimulated; "recurrent', animals which were stimulated twice a day for a week. Body weight is amount gained over the week of testing. Food and water intake are the average daily values over this period. Basal thresholds and core temperature were taken at the end of testing on the day prior to sacrifice. See Methods for details. Mean + S.E.M. given. F a n d P values are derived from a one-way analysis of variance.

Body weight (g)

Adrenal weight (g)

Food intake (g/day)

Water intake (g/day)

Core Basal nociceptive threshold temperature Heat (s) Prersurc"~ (°C)

Home (n = 6)

40.01 + 2.08

58.91 + 0.79

21.67 + 0.89

47.33 + 3,22

37.86 + 0.09

3.79 + 0,07

124.92 +_ 8.16

Chamber (n = 8)

37.05 + 0.81

55.81 -+ 3.66

22.87 + 0.81

42.37 + 2.99

37,87 _+ 0.06

3.83 + 0.06

126.86 _+ 12.10

Recurrent (~z = 10)

36.75 + 1.51

57.16 + 0.65

20.88 + 1.02

45.?'.1± 3.35

37.84 _+ 0.05

3.92 + 0.06

120.26 + 6.80

~.2~

1.92

0.51

0.31

0.44

0.01

0.92

1.27

P

0.17

0.58

0.74

0.65

0.99

0.42

0.3 !

Influence o f repeated stimulation upon physiological and behavioural parameters and opioid peptide sys-

of stimulation. A doubling of the current intensity on

tems

ficacy a n d a s t e p - w i s e e l e v a t i o n in c u r r e n t i n t e n s i t y

s e s s i o n 5 r e s u l t e d in an i n c r e a s e in a n t i n o c i c e p t i v e ef-

A s s h o w n in Fig. 6, a s i g n i f i c a n t ' t o l e r a n c e " d e v e l -

o v e r t h e r e m a i n i n g test p e r i o d a l l o w e d for a m a i n t e -

oped within 4 sessions to the antinociceptive effects

n a n c e o f a s t a b l e level o f a n t i n o c i c e p t i o n . T h e r e w a s

,,==.w

160"

0

o q?== II

w

140

0 rU'~ t__ r-

120

• ¢D

Heal Pressure

0

o~

100 ,I,.l'. •

;

1"1

G

1}.

2.0

2.25

SESSION ( 2 / d a y ) •

0.30 0.30

0.30

0.30

0.60

|

0.70

O.BO

0.90

1.0

1.25

1.50

1.75

CURRENT. (mAMP) Fig. 6. Maintenance of the antinociceptive effects of stimulation by stcp-wise increase in current intensity. Rats were stimulated at :m intensity of 3001tA for the first 4 sessions until a degree of tolerance had developed: thereafter current was sequentially increased for each of the remaining 10 sessions. For further details, see Methods. Mean ± S.E.M. shown, n = 10. Asterisks refer to the significance of values on session 4 vs session 1, and on session 5 vs session ,1 (***P ~ 0.001, paired t-test).

1116 no significant difference between the antinociception elicited at a current intensity of 2.25 mA on the fourteenth session and that of 300,uA on the first session. On no session did rats placed in the chamber and not stimulated show any antinociception (not shown). Rats which were stimulated for the firs~ time on session 14 showed a full antinociception: for heat, 159.77 + 8.3% and for pressure, 147.6.4- 7.1%. On the previous 13 sessions on which they were placed in the chamber and not stimulated, they had shown no antinociception (not shown). From Table II, it is clear that there were no differences between the groups as regards basal nociceptire thresholds and core temperature, Further, the body weight gained across the 7 days of experimentation and the average food and water intake throughout tliis period were not modified by recurrent stimulation, In addition, adrenal weights were found not to differ between any of the groups (Table II) (values for rats stimulated only on session 14, not shown). In systemic plasma, no significant difference could be seen amongst the various groups in the levels of immunoreactive (ir)-fl-EP or ir-PRL, though in the case of PRL, there was a tendency towards an in-

crease in recurrently stimulated animals. In both the anterior and neurointermediate lobes of the pitoitarv, as compared to animals which had remained at home, all other groups revealed a rise in levels of irfl-EP (Table III). Ir-DYN was affected in neither of these lobes. In neither the midbrain PAG (Table 11I) nor any other brain structure examined (not shown) could any significant alteration be seen in levels of irfl-EP between the various groups examined. In addition, in discrete structures of brain and in the spinal cord, there were no significant differences in the levels of either ir-Met-enkephalin or ir-DYN (not shown). DISCUSSION The present study demonstrates that the SPA elicited from the ventral midbrain PAG of the freely moving rat displays tolerance: that is, upon recurrent stimulation, there is a shift to the right of the d o s e response (current intensity-antinociception) relationship. This is comparable to the tolerance which develops upon repeated administration of endogenous or exogenous opioids such as fl-EP or morphine

TABLE I!1

The in/h~enee of recurrent electrical stimulatiot~ of the nzidbrain periaqueductal grey upon discrete tissue levels of immunoreaetive fl-en. dorphin (ir.fl.EPL im,mnoreactive prolactit: (ir.PRL), itnmunoreactive dynorphit~ (ir.DYN) attd inmmnoreactive Met.enkephalin Or.ME) 'ltome' signifiesanimals which remained in their home cages; chamber, animals which were placed in the chamber twice a day Ik~ra week but not stimulated;'single', rats which were treated likewisebut stimulatedonly on the final session; 'recurrent', rats which were stimulated on each session. For further details, see Methods. Levels are expressed either per milliliter (plasma) or per milligramwet weight. Mean _+S.E.M. given. F and P values are derived from a one.way analysisof variance, A Student-Newman-Keuls analysis revealed that 'home' animalswere significantly(P ~ 0.05) different from the other groups as regards levels of ir-/3-EP in both the anterior and neurointermediatepituitary,

Plasma

Anterior lobe

Neuroimermediate lobe

Midbrain periaqueducml grey ir.~.EP (finollmg)

ir.fl-EP (fmollmi)

ir.PRL (nglml)

ir.fl.EP (pmollmg)

ir.DYN (fmollmg)

ir.fl-EP ir.fl-DYN ~,pmollmg) tfinolimg)

ir-DYN (fmollmg)

Jr-ME (fmol/mg)

Home (n = 6)

45.06+ 13.06

3,46_+ 0,80

49,18+ 2,78

295.54_+ 24.00

411.70_+ 21.88

1121,11+ 2.58_+ 140,17 0,34

18.16+ 2,07

541.19+ 14.08

Chamber (n = 8)

38,74_+ 11.78

3,28+ 1,05

60.67+ 3,44

258.01+ 22,09

559.44-446,29

1339,16+ 2,65+ 151,09 0,33

18,20+ 1.77

521.99+ 41.66

Single (n = 7)

63.24_+ 21.49

2,51 + 0,86

67,83_+ 4,59

354,56+ 43,94

543.22+ 43.29

1230,61+ 116, I 1

3,24+ 0,31

19,14-+ 2.77

533,72_ 35.12

Recurrent (n = 10)

55,00_+, 12.,19.

5,42_+ 1,32

64,43+ 3,39

259,64_+ 28.06

599,18-+ 43,11

1260,00-+ 2,83+ 30,88 0,41

15,07+ 1.89

506.11441.22

F~.,.~

11.88

1,48

3,50

1,51

3,93

0,59

0,71

0,89

0.59

P

0.46

0,24

0,03

0,23

0.02

0,63

0.56

0.46

0.63

107 and is in line with the mediation of this form of SPA by an opioidergic mechanism, indeed, a bilateral cross-tolerance is apparent between SPA elicited from the PAG and morphine 24"35"37"38''~3. M o ~ h i n e acts predominantly at ~t-reeeptors and our previous data indicate that F~-receptors mediate the SPA which can be elicited from the PAG 43. The ability of a low dose of naloxone (selective for/~-receptors) not only to block SPA in naive rats 43 but also its reinstatement in tolerant rats suggests that this reinstated SPA is similarly exerted via/~-receptors. Further, the parallelism betweeJ~ the dose-response curves for SPA in naive and tolerant rats is also consistent with a mediation of this reinstated SPA by the same population of 0a) receptors. Evidently, the tolerance which is displayed to SPA from the PAG closely conforms to the pharmacological understanding of this phenomenon. In contrast to the antinociception, neither tae hyperthermia nor locomotor effects of stimulation are mediated by opioids 4°'43. In the case of the hyperthermia, no indication of a 'tolerance' was ~een. As regards the locomotor effects, the data were equivocal in that their intensity remained constant whereas the threshold for their initiation progressively rose. A discussion of mechanisms is difficult in that the neuronal substrate mediating these effects is not definitely known. They po:isibly reflect, however, a direct stimulation of the serotoninergic pathway from the dorsal rapht~ which is inhibitory to the nigrostriatal dopaminergic projection '''~'17'47. Thu,,~, it may bc that the locomotor effects are ultimately dopaminergic in nature. Notably. the effects of recurrent stinaulation of dopamine receptors on motor behaviour are not well understood and there are reports of both tolerance and sensitization 23"27as'5". As regards the antinociceptive effects of stimulation, although it is clear that a tolerance does develop, it is also apparent that the mechanisms underlying this tolerance might not necessarily be the same as those responsible for tolerance to the adminisnation of opioids such as morphine. There are a number of possibilities. Firstly, the loss of SPA could be non-specific. For example, recurrent stimulation may have caused local tissue damage. This possibility cannot be completely discounted but is unlikely to be the predominant factor underlying tolerance. Thus, we specifical-

ly examined electrode locations and could see no evidence of tissue destruction other than a local gliosis also seen in unstimulated rats with chronic electrodes. Further, animals recovered from tolerance which does not occur in animals sustaining small lesions at the electrode site 37. Moreover, a local lesion is very unlikely to explain the cross-tolerance seen to morphine 43. In addition it might be argued that the rats are generally debilitated such that they were incapable of generating a full antinociception. This argument is strongly countered by the following points. Thus, the SPA can be reinstated by an increase in current intensity, tolerant rats show a full antinociceptive response to a ~:-opioid agonist a3 and the rats are in excellent condition, revealing no alterations in any of the stress-sensitive parameters evaluated. Secondly, recurrent stimulation may somehow deplete or incapacitate the opioid peptide network mediating SPA. We previously suggested that it is an activation of pools of fl-EP intrinsic to the PAG which underlies the SPA evoked therefrom and acute stimulation results in a rapid diminution in PAG levels of ir-fl-EP reflecting its release 4~'~4. Twenty-four hours post-stimulation, recurrently stimulated rats revealed no alteration in steady-state levels of i1-fl-EP either in the PAG or the hypothalamus, the perikaryal origin of PAG pools of fl-EP 16. In addition, we detected no change in levels of ir-Met-enkephalin or ir-DYN in the PAG or various discrete regions of brain and spinal cord. Further, the behavioural, physiological and endocrinological parameters monitored are without exception sensitive to control by opioidergic mechanisms (and specifically by/3.EP) and manifested no indication of any disturbance in their functional activity. Moreover, a sustained depression in endogenous opioid activity should lead to a supersensitivity of opioid receptors to opioid agonists 32"43"63, and this is not the case 43. Thus, it is unlikely that recurrent stimulation led to a decrease in the activity of the endogenous opioid peptide (probably /~-EP) mediating SPA and that this resulted in an apparent tolerance. In fact, it was interesting to note that there was no induction of/3-EP in the PAG. An increased storage (synthesis and release) of particular opioid peptides has been seen upon repeated stimulation by various nlanipulations t~''~'ata2a~'a~' 53.0,. Conceivably, for such an event it is essential that the perikarya wherein the pcptide is swlthesized be

1(18 directly stimulated whereas in the present case it is the fibres and terminals which are being excited. Moreover. neither of the above explanations of tolerance could account for the occurrence of a reduction in the antinociceptive efficacy of morphine in rats rendered tolerant to stimulation 38'43. This observation provides powerful evidence for the occurrence of tolerance at the level o~ (~t-) opioid receptors and coupled mechanisms. Thus, the most reasonable hypothesis is that recurrent stimulation is associated with the repeated activation and release of an endogenous opioid peptide acting at opioid receptors: this leads to a tolerance in a tashion analogous to that seen upon repeated application of morphine. If our previous contention ~°'aa that PAG pools of fl-EP mediate the antinociception is correct, then the (~-) receptor at which tolerance develops should be in the PAG itself: this argument is supported by the observation that the antinociceptive action of morphine applied directly into the PAG is reduced in rats tolerant to stimulation elicited therefrom 38. However, the location of the receptor cannot, presently, be specified with certainty. Further, it is also possible that tolerance develops in a postopioid receptor mechanism involved in the expression of the antinociceptive actions of (/~-) opioids, For example, serotonin has been implicated in this respect and evidence that tolerance can develop upon repetitive activation of serotonin nuclei has been presented by Oliveras et al. ~'~, Nonetheless, in addition to cellular and molecular bases of tolerance, situational, psychological factors may be of significance. Tolerance develops more rapidly to morphine and is most pronounced upon presentation of the drug and testing of the animals in a predictable environment accompanied by constant cues-'.4A3,1s2s,.~a,5,',,sr, It is possible that associational' learning processes could have contributed to the tolerance which developed to stimulation of the PAG. That such factors did indeed play a role cannot be excluded and it was of importance to specifically address the possibility that a 'conditioned tolerance' could completely explain the present findings. In the seventies, Siegel formulated the hypothesi,,{that 'paviovian conditioning' can explain the tolerance which develops to repeated application of morphine 55,5~', It is supposed that cues (CS) associated with the administration of the drug (UCS) gradually acquire the

ability to elicit a conditioned response (CR) opposite in direction to that produced by the drug itself (UCR): that is, this CR effectively cancels the action of the drug and this process is reflected in the development of tolerance. A central prediction of the "pavlovian hypothesis" is that upon exposure to these cues (the CS) in the absence of the drug (the UCS), the CR should be elicited. In the case of morphine or stimulation of the PAG, this CR would obviously be a hyperalgesia. However, exposure to all cues associated with stimulation failed to produce such a hyperalgesia. This finding strongly argues against such an account of the development of tolerance to PAG stimulation. A second tenet of this conditioning model is that tolerance should be only extremely slow to recover spontaneously (or not recover at all) in the absence of extinction trials, that is of repetitive presentation of th~ cues associated with stimulation (CS). Repeated exposure to these cues should accelerate the loss of tolerance. However, this was found not to be the case in the present study, similarly in contradiction with this account of tolerance. This finding also tends to refute the possibility that any form of conditioning (as conceptualized in more recent, sophisticated theories 4't3"1~'57) plays a major role in the development of tolerance. Nevertheless, that there may be some form of learning taking place is revealed by the (albeit mild) antinociception shown by tolerant rats re-exposed to the cues accompanying stimulation (Fig. 3). Interestingly, rats stimulated on this occasion showed no antinociception: possibly stimulation might come to activate a hyperalgesic process or somehow interfere with the appearance of the conditioned antinociception. This mild antinociception rapidly decayed upon repeated exposure to these cues, that is it showed extinction. This observation is of particular interest in the light of studies in which, rather than a CR opposite to the effects of morphine, a CR acting similarly to morphine has been seen - - a mechanism proposed to explain the sensitization which occurs to certain effects of morphine upon its repeated application in low doses at large time intervals ~3,57. Further, this conditioned antinociception bears direct comparison with that which is mediated by opioids and can be conditioned upon repeated presentation of both rewarding and aversive stimuli I~j.13'2~.62.Indeed, the antinociceptive efficacy of noxious stressors has been

109 shown to rapidly adapt and those cues associated with its imposition to acquire thc ability themselves to evoke an antinociception t°a3"-'°'62. Directly analogous to the prescnt data, this similarity remforces previous contentions that there are certain communalities between SPA and the antinociception elicited by stress as regards the underlying neuronal substrates and mechanisms~'~t. However, it is important to emphasize that the effects of brain stimulation can be clearly differentiated from those 6f stress. Our previous biochemical, pharmacological and behavioural studies of acute PAG stimulation have clearly distinguished its effects from those of acute stress 19"~°m-4~. Further, as detailed in our prior publications 4°'43"44, we see absolutely no signs of aversion in rats stimulated in this region of the ventral PAG: rats never vocalize, attempt to escape, appear distressed and are not aggressive following stimulation. This lack of aversive effects is entirely in agreement with previous work identifying the dorsal PAG as the region the stimulation of which is aversive t~as'29`36`st. The present data with chronic stimulation support the contention that stimulation of the ventral PAG is not aversive and does not represent a stress: evidently, SPA is not just a form of stress-induced antinociception. In contrast to the influence of chronic stress upon basal nociceptive thresholds, ingestive behaviour, body weight, core temperature attd adrenal weights 3,tt''t'26`3~'41.53.'~4.58 none of these variables were affected by recurrent stimulation of the PAG. Chronic stress also exerts a pronounced influence upon certain CNS and hypophyseal pools of opioid peptides, in particular spinal cord pools of D ~ N ar.d adenohypophyseal pools of fl-EP 3,21,31,42,45"53. In nt,merous discrete regions of brain, spinal cord and pituitary, no specific effects of

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repetitive brain stimulation could be seen. The only chang~ detected was the rise in levels of ir-fl-EP in both lobes of the pituitary: this was not particular to stimulation but common to all ~ats placed in the chamber and presumably reflects the mild stress of novelty, handling and testing, etc. The ability of acute stimulation to elevate circulating levels of irPRL also does not constitute a stress-effect but a direct excitation of serotoninergic neurones in the dorsal raph6, which play a facilitatory role in the control of PRL secretion 1"26"31"46"52"~1.The dorsal raph6 does not, in fact, play a role in the modulation of the response of lactotrophs to stress 61. It is thus of interest that while chronic exposure to stress usually fails to affect or decreases levels of PRL in systemic plasma tt'26"45, in repetitively stimulated animals, no significant increase was seen. Thus, it appears that the stimuli of either stress or PAG stimulation, which are mediated by different mechanisms and both of which elicit a striking enhancement of PRL secretion when acutely applied, do not greatly modify basal secretion when presented chronically. In conclusion, the major finding of this study is that a genuine tolerance develops upon recurrent antinociceptive stimulation of PAG. An analysis of the mechanisms which could underlie this tolerance suggest that this probably occurs at the levels of the receptor and coupled mechanisms, thereby explaining the cross-tolerance seen to morphine in rats rendered tolerant to the SPA elicited from the PAG. ACKNOWLEDGEMENTS We would like to thank A. Huber for assistance with the biochemical assays. This study was supported by the Deutsche Forschungsgemeinschaft.

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